Cytology and cell-block immunohistochemistry of circulating tumour cells.

OBJECTIVE: The study set out to assess the feasibility of using ParsortixTM circulating tumour cell (CTC) extraction and CytoFoam Disc cell-block immunohistochemistry to diagnose metastatic carcinoma from blood samples in a National Health Service district general hospital. METHODS: Blood samples were taken from 50 patients with metastatic carcinoma and 50 healthy volunteers and processed, using a previously published method, to extract CTCs and collect them in a cell-block for routine formalin-fixed paraffin sectioning and immunohistochemistry. The extracted cells were compared with the patients' routine diagnostic samples. RESULTS: The samples from the 50 carcinoma patients showed cytokeratin-positive cells in 19 cases. In eight of these, the cytokeratin-positive cells had a similar immunoprofile to the carcinoma in the conventional biopsy or cytology specimen. Some carcinoma patients also had circulating cytokeratin-positive cells that were probably benign epithelial cells and circulating megakaryocytes. Both of these types of cells were also found in healthy volunteers. Processing and initial examination could be completed in 2 days. The full processing cost was approximately £316 per case. CONCLUSIONS: CTCs could be extracted from the blood of some patients with metastatic carcinoma and formed into a formalin-fixed cell-block for routine paraffin processing and immunohistochemistry. The specificity of this approach is constrained by the observation that some patients with metastatic carcinoma had circulating cytokeratin-positive cells that were probably benign, and these were also found in healthy volunteers. Circulating megakaryocytes were present in carcinoma patients and healthy volunteers.

Differential transcription factor expression by human epithelial cells of buccal and urothelial derivation.

Identification of transcription factors expressed by differentiated cells is informative not only of tissue-specific pathways, but to help identify master regulators for cellular reprogramming. If applied, such an approach could generate healthy autologous tissue-specific cells for clinical use where cells from the homologous tissue are unavailable due to disease. Normal human epithelial cells of buccal and urothelial derivation maintained in identical culture conditions that lacked significant instructive or permissive signaling cues were found to display inherent similarities and differences of phenotype. Investigation of transcription factors implicated in driving urothelial-type differentiation revealed buccal epithelial cells to have minimal or absent expression of PPARG, GATA3 and FOXA1 genes. Retroviral overexpression of protein coding sequences for GATA3 or PPARy1 in buccal epithelial cells resulted in nuclear immunolocalisation of the respective proteins, with both transductions also inducing expression of the urothelial differentiation-associated claudin 3 tight junction protein. PPARG1 overexpression alone entrained expression of nuclear FOXA1 and GATA3 proteins, providing objective evidence of its upstream positioning in a transcription factor network and identifying it as a candidate factor for urothelial-type transdifferentiation or reprogramming.

Heterarchy of transcription factors driving basal and luminal cell phenotypes in human urothelium.

Cell differentiation is affected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq was used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq, as having altered accessibility during differentiation, were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including forkhead box A1 (FOXA1), P63, GRHL2, CTCF and GATA-binding protein 3 (GATA3). In addition, co-occurrence of GATA3 motifs was observed within subsets of differentiation-specific peaks containing P63 or FOXA1. Changes in abundance of GRHL2, GATA3 and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally regulated programme, urothelial differentiation operates as a heterarchy, wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes.